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目的:研究异常甲基化在调控结直肠癌细胞中CES2基因表达中的作用,并观察结直肠癌细胞内CES2表达与化疗敏感性的关系。方法:采用MSP检测八种结直肠癌细胞系中CES2基因启动子甲基化;荧光定量PCR和westernblot方法检测DAC去甲基化后CES2 mRNA及蛋白表达变化;采用MTT法,检测各型结肠癌细胞株对伊利替康的细胞毒性,计算IC50并分析其与CES2 mRNA表达的相关性。结果:CES2基因启动子在八种结直肠细胞株中均呈未甲基化状态,使用DAC处理后,CES2在其低表达的细胞株无明显增加,不同结直肠癌细胞株对CPT-11敏感性存在差异(P<0.05),其中LoVo细胞对CPT-11敏感性最低,RKO细胞对CPT-11的敏感性最高。结直肠细胞株内CES2表达与IC50呈负相关性(r=-0.58651,P<0.01)。结论:甲基化不参与调控CES2基因的表达,CES2表达与CPT-11细胞毒性呈正相关。
OBJECTIVE: To investigate the role of abnormal methylation in the regulation of CES2 gene expression in colorectal cancer cells and to observe the relationship between CES2 expression and chemosensitivity in colorectal cancer cells. Methods: Methylation of CES2 promoter in eight colorectal cancer cell lines was detected by MSP. The changes of CES2 mRNA and protein expression after demethylation of DAC were detected by fluorescence quantitative PCR and western blot. MTT assay was used to detect the expression of various types of colon cancer Cell lines were tested for cytotoxicity to irinotecan, IC50 was calculated and analyzed for correlation with CES2 mRNA expression. Results: The CES2 gene promoter was unmethylated in all the eight colorectal cell lines. After treatment with DAC, CES2 showed no significant increase in its low expression of cell lines, and different colorectal cancer cell lines were sensitive to CPT-11 (P <0.05). Among them, LoVo cells had the lowest sensitivity to CPT-11 and RKO cells had the highest sensitivity to CPT-11. CES2 expression in colorectal cancer cells was negatively correlated with IC50 (r = -0.58651, P <0.01). Conclusion: Methylation is not involved in the regulation of CES2 gene expression. CES2 expression is positively correlated with CPT-11 cytotoxicity.